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• W2034151760 abstract "Ubiquitination of murine cyclin E is triggered by phosphorylation on threonine 393. Cyclin ET393A knockin mice exhibited increased cyclin E stability, but no phenotypic abnormalities. Importantly, loss of the p53 pathway exacerbated the effect of the T393A mutation. Thus, in p21−/− cells the T393A mutation had an exaggerated effect on cyclin E abundance and its associated kinase activity, which caused abnormal cell cycle progression, and genetic instability involving chromosome breaks and translocations. Moreover, cyclin ET393A acted synergistically with p53 deficiency to accelerate tumorigenesis in cyclin ET393A p53−/− mice; Ras more readily transformed cyclin ET393A p53−/− cells than p53−/− cells in vitro; and cyclin ET393A mice had a greatly increased susceptibility to Ras-induced lung cancer. Ubiquitination of murine cyclin E is triggered by phosphorylation on threonine 393. Cyclin ET393A knockin mice exhibited increased cyclin E stability, but no phenotypic abnormalities. Importantly, loss of the p53 pathway exacerbated the effect of the T393A mutation. Thus, in p21−/− cells the T393A mutation had an exaggerated effect on cyclin E abundance and its associated kinase activity, which caused abnormal cell cycle progression, and genetic instability involving chromosome breaks and translocations. Moreover, cyclin ET393A acted synergistically with p53 deficiency to accelerate tumorigenesis in cyclin ET393A p53−/− mice; Ras more readily transformed cyclin ET393A p53−/− cells than p53−/− cells in vitro; and cyclin ET393A mice had a greatly increased susceptibility to Ras-induced lung cancer. The abundance of cyclin E is increased in many human tumors and is predictive of tumor aggression and clinical outcome. However, it has been uncertain whether elevated cyclin E is a downstream marker for aggressive tumors or represents a driving force behind tumor progression. Our mouse model strongly supports the latter, by showing that inactivation of the normal pathway for cyclin E degradation, by direct mutation of the cyclin E gene itself, disrupts cell cycle regulation, causes genetic instability, and is, per se, oncogenic. Moreover, our mouse model shows that the impact of this misregulated cyclin E is constrained by the p53-p21 pathway, such that simultaneous loss of p53 and misregulation of cyclin E cooperate to cause oncogenic cell transformation. Eukaryotes express regulatory proteins, called cyclins, which are required for the cell division cycle and function primarily by binding to and allosterically activating cyclin-dependent kinases (CDKs) (Sherr and Roberts, 1999Sherr C.J. Roberts J.M. CDK inhibitors: positive and negative regulators of G1-phase progression.Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (4936) Google Scholar). Cyclin E1 associates predominantly with one member of the CDK family, Cdk2, and serves a dual purpose in activating cell proliferation: it phosphorylates and thereby inhibits proteins that impede cell cycle progression (p27Kip1, pRb), and it phosphorylates and thereby activates proteins that promote cell division (Hwang and Clurman, 2005Hwang H. Clurman B. Cyclin E in normal and neoplastic cell cycles.Oncogene. 2005; 24: 2776-2786Crossref PubMed Scopus (333) Google Scholar, Sherr and Roberts, 2004Sherr C.J. Roberts J.M. Living with or without cyclins and cyclin-dependent kinases.Genes Dev. 2004; 18: 2699-2711Crossref PubMed Scopus (850) Google Scholar). These functions of cyclin E are essential when nondividing cells exit the quiescent state and resume cell proliferation but may be redundant with the activities of other cell cycle regulators in continuously proliferating cells (Geng et al., 2003Geng Y. Yu Q. Sicinska E. Das M. Schneider J.E. Bhattacharya S. Rideout W.M. Bronson R.T. Gardner H. Sicinski P. Cyclin E ablation in the mouse.Cell. 2003; 114: 431-443Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar). Cyclin E expression and its associated kinase activity are periodic and reach a peak in early S phase (Koff et al., 1992Koff A. Giordano A. Desai D. Yamashita K. Harper J.W. Elledge S. Nishimoto T. Morgan D.O. Franza B.R. Roberts J.M. Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle.Science. 1992; 257: 1689-1694Crossref PubMed Scopus (891) Google Scholar). This periodicity is regulated by E2F-dependent cyclin E transcription (Botz et al., 1996Botz J. Zerfass-Thome K. Spitkovsky D. Delius H. Vogt B. Eilers M. Hatzigeorgiou A. Jansen-Durr P. Cell cycle regulation of the murine cyclin E gene depends on an E2F binding site in the promoter.Mol. Cell. Biol. 1996; 16: 3401-3409Crossref PubMed Scopus (223) Google Scholar, Geng et al., 1996Geng Y. Eaton E.N. Picon M. Roberts J.M. Lundberg A.S. Gifford A. Sardet C. Weinberg R.A. Regulation of cyclin E transcription by E2Fs and retinoblastoma protein.Oncogene. 1996; 12: 1173-1180PubMed Google Scholar, Ohtani et al., 1995Ohtani K. DeGregori J. Nevins J.R. Regulation of the cyclin E gene by transcription factor E2F1.Proc. Natl. Acad. Sci. USA. 1995; 92: 12146-12150Crossref PubMed Scopus (513) Google Scholar) and ubiquitin-mediated cyclin E proteolysis (Clurman et al., 1996Clurman B.E. Sheaff R.J. Thress K. Groudine M. Roberts J.M. Turnover of cyclin E by the ubiquitin-proteasome pathway is regulated by cdk2 binding and cyclin phosphorylation.Genes Dev. 1996; 10: 1979-1990Crossref PubMed Scopus (402) Google Scholar, Won and Reed, 1996Won K.A. Reed S.I. Activation of cyclin E/CDK2 is coupled to site-specific autophosphorylation and ubiquitin-dependent degradation of cyclin E.EMBO J. 1996; 15: 4182-4193Crossref PubMed Scopus (302) Google Scholar). Ectopic overexpression of cyclin E causes cell cycle anomalies and genetic instability (Ohtsubo and Roberts, 1993Ohtsubo M. Roberts J.M. Cyclin-dependent regulation of G1 in mammalian fibroblasts.Science. 1993; 259: 1908-1912Crossref PubMed Scopus (652) Google Scholar, Spruck et al., 1999Spruck C.H. Won K.A. Reed S.I. Deregulated cyclin E induces chromosome instability.Nature. 1999; 401: 297-300Crossref PubMed Scopus (591) Google Scholar, Minella et al., 2002Minella A.C. Swanger J. Bryant E. Welcker M. Hwang H. Clurman B.E. p53 and p21 form an inducible barrier that protects cells against cyclin E-cdk2 deregulation.Curr. Biol. 2002; 12: 1817-1827Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). It is therefore thought to be critical for normal cells to tightly regulate cyclin E activity; indeed, increased cyclin E activity is a hallmark of cancer cells. Two ubiquitination pathways promote cyclin E degradation. Monomeric cyclin E (not bound to cdk2) is targeted by a Cullin-3 ubiquitin ligase (Singer et al., 1999Singer J.D. Gurian-West M. Clurman B. Roberts J.M. Cullin-3 targets cyclin E for ubiquitination and controls S phase in mammalian cells.Genes Dev. 1999; 13: 2375-2387Crossref PubMed Scopus (315) Google Scholar). However, cyclin E that associates with Cdk2 does not interact with Cullin-3 and instead is degraded by a pathway involving the SCFFbw7 ubiquitin-protein ligase (Strohmaier et al., 2001Strohmaier H. Spruck C.H. Kaiser P. Won K.A. Sangfelt O. Reed S.I. Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line.Nature. 2001; 413: 316-322Crossref PubMed Scopus (480) Google Scholar, Koepp et al., 2001Koepp D.M. Schaefer L.K. Ye X. Keyomarsi K. Chu C. Harper J.W. Elledge S.J. Phosphorylation-dependent ubiquitination of cyclin E by the SCFFbw7 ubiquitin ligase.Science. 2001; 294: 173-177Crossref PubMed Scopus (621) Google Scholar, Moberg et al., 2001Moberg K.H. Bell D.W. Wahrer D.C. Haber D.A. Hariharan I.K. Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancer cell lines.Nature. 2001; 413: 311-316Crossref PubMed Scopus (366) Google Scholar). Fbw7 is a bifunctional adaptor that brings phosphorylated cyclin E into proximity with the remainder of the SCF complex. At least four cyclin E phosphorylation sites regulate Fbw7-driven cyclin E ubiquitination; in human cyclin E these are T62, S372, T380, and S384 (Welcker et al., 2003Welcker M. Singer J. Loeb K.R. Grim J. Bloecher A. Gurien-West M. Clurman B.E. Roberts J.M. Multisite phosphorylation by Cdk2 and GSK3 controls cyclin E degradation.Mol. Cell. 2003; 12: 381-392Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Most important is T380, which is phosphorylated by both GSK3 and Cdk2 itself. T380 falls within a canonical Fbw7 recognition motif, and T380 phosphorylation allows Fbw7 to bind to cyclin E (Orlicky et al., 2003Orlicky S. Tang X. Willems A. Tyers M. Sicheri F. Structural basis for phosphodependent substrate selection and orientation by the SCFCdc4 ubiquitin ligase.Cell. 2003; 112: 243-256Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, Ye et al., 2004Ye X. Nalepa G. Welcker M. Kessler B. Spooner E. Qin J. Elledge S.J. Clurman B.E. Harper J.W. Recognition of phosphodegron motifs in human cyclin E by the SCFFbw7 ubiquitin ligase.J. Biol. Chem. 2004; 279: 50110-50119Crossref PubMed Scopus (109) Google Scholar). Deregulated cyclin E activity is thought to play a fundamental role in tumorigenesis. A cyclin E transgene that overexpresses cyclin E induces breast carcinomas in mice, and ectopic cyclin E overexpression both induces genetic instability in cultured cells and transforms rodent fibroblasts in combination with other oncogenes (Bortner and Rosenberg, 1997Bortner D.M. Rosenberg M.P. Induction of mammary gland hyperplasia and carcinomas in transgenic mice expressing human cyclin E.Mol. Cell. Biol. 1997; 17: 453-459Crossref PubMed Scopus (196) Google Scholar, Minella et al., 2002Minella A.C. Swanger J. Bryant E. Welcker M. Hwang H. Clurman B.E. p53 and p21 form an inducible barrier that protects cells against cyclin E-cdk2 deregulation.Curr. Biol. 2002; 12: 1817-1827Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, Spruck et al., 1999Spruck C.H. Won K.A. Reed S.I. Deregulated cyclin E induces chromosome instability.Nature. 1999; 401: 297-300Crossref PubMed Scopus (591) Google Scholar). Cyclin E containing a mutation at T380 that prevents its recognition by Fbw7 was more potent than wt-cyclin E with respect to causing cell cycle deregulation and genetic instability. Many studies have shown that high cyclin E protein expression in human cancers is associated with increased tumor aggressiveness and poor patient outcome (Keyomarsi et al., 2002Keyomarsi K. Tucker S.L. Buchholz T.A. Callister M. Ding Y. Hortobagyi G.N. Bedrosian I. Knickerbocker C. Toyofuku W. Lowe M. et al.Cyclin E and survival in patients with breast cancer.N. Engl. J. Med. 2002; 347: 1566-1575Crossref PubMed Scopus (481) Google Scholar, Porter et al., 1997Porter P.L. Malone K.E. Heagerty P.J. Alexander G.M. Gatti L.A. Firpo E.J. Daling J.R. Roberts J.M. Expression of cell-cycle regulators p27Kip1 and cyclin E, alone and in combination, correlate with survival in young breast cancer patients.Nat. Med. 1997; 3: 222-225Crossref PubMed Scopus (833) Google Scholar, Dong et al., 2000Dong Y. Sui L. Tai Y. Sugimoto K. Hirao T. Tokuda M. Prognostic significance of cyclin E overexpression in laryngeal squamous cell carcinomas.Clin. Cancer Res. 2000; 6: 4253-4258PubMed Google Scholar, Fukuse et al., 2000Fukuse T. Hirata T. Naiki H. Hitomi S. Wada H. Prognostic significance of cyclin E overexpression in resected non-small cell lung cancer.Cancer Res. 2000; 60: 242-244PubMed Google Scholar, Hwang and Clurman, 2005Hwang H. Clurman B. Cyclin E in normal and neoplastic cell cycles.Oncogene. 2005; 24: 2776-2786Crossref PubMed Scopus (333) Google Scholar). In a few cancers, the cyclin E gene is amplified, but in most cases cyclin E deregulation may result from the disruption of pathways that govern its synthesis and its destruction. Fbw7 mutations have been described in cancer lines that contain high amounts of cyclin E protein as well as in primary cancers (Spruck et al., 2002Spruck C.H. Strohmaier H. Sangfelt O. Muller H.M. Hubalek M. Muller-Holzner E. Marth C. Widschwendter M. Reed S.I. hCDC4 gene mutations in endometrial cancer.Cancer Res. 2002; 62: 4535-4539PubMed Google Scholar, Strohmaier et al., 2001Strohmaier H. Spruck C.H. Kaiser P. Won K.A. Sangfelt O. Reed S.I. Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line.Nature. 2001; 413: 316-322Crossref PubMed Scopus (480) Google Scholar, Moberg et al., 2001Moberg K.H. Bell D.W. Wahrer D.C. Haber D.A. Hariharan I.K. Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancer cell lines.Nature. 2001; 413: 311-316Crossref PubMed Scopus (366) Google Scholar, Calhoun et al., 2003Calhoun E.S. Jones J.B. Ashfaq R. Adsay V. Baker S.J. Valentine V. Hempen P.M. Hilgers W. Yeo C.J. Hruban R.H. Kern S.E. BRAF and FBXW7 (CDC4, FBW7, AGO, SEL10) mutations in distinct subsets of pancreatic cancer: potential therapeutic targets.Am. J. Pathol. 2003; 163: 1255-1260Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Mouse models have confirmed that Fbw7 is a tumor suppressor gene (Rajagopalan et al., 2004Rajagopalan H. Jallepalli P.V. Rago C. Velculescu V.E. Kinzler K.W. Vogelstein B. Lengauer C. Inactivation of hCDC4 can cause chromosomal instability.Nature. 2004; 428: 77-81Crossref PubMed Scopus (453) Google Scholar, Mao et al., 2004Mao J.H. Perez-Losada J. Wu D. Delrosario R. Tsunematsu R. Nakayama K.I. Brown K. Bryson S. Balmain A. Fbxw7/Cdc4 is a p53-dependent, haploinsufficient tumour suppressor gene.Nature. 2004; 432: 775-779Crossref PubMed Scopus (297) Google Scholar). Although these data support a role for cyclin E in human tumorigenesis, the effects of cyclin E stabilization in general, and the specific role of cyclin E deregulation in Fbw7-associated tumorigenesis remain unclear. On the one hand, the biological effects of cyclin E stabilization have only been examined in settings that also involve ectopic cyclin E overexpression. On the other hand, Fbw7 also targets c-Myc, c-Jun, and Notch for degradation, and each of these proteins are themselves oncogenes when overexpressed (Welcker et al., 2004Welcker M. Orian A. Jin J. Grim J.A. Harper J.W. Eisenman R.N. Clurman B.E. The Fbw7 tumor suppressor regulates glycogen synthase kinase 3 phosphorylation-dependent c-Myc protein degradation.Proc. Natl. Acad. Sci. USA. 2004; 101: 9085-9090Crossref PubMed Scopus (611) Google Scholar, Nateri et al., 2004Nateri A.S. Riera-Sans L. Da Costa C. Behrens A. The ubiquitin ligase SCFFbw7 antagonizes apoptotic JNK signaling.Science. 2004; 303: 1374-1378Crossref PubMed Scopus (287) Google Scholar, Wu et al., 2001Wu G. Lyapina S. Das I. Li J. Gurney M. Pauley A. Chui I. Deshaies R.J. Kitajewski J. SEL-10 is an inhibitor of notch signaling that targets notch for ubiquitin-mediated protein degradation.Mol. Cell. Biol. 2001; 21: 7403-7415Crossref PubMed Scopus (269) Google Scholar). Thus, the neoplasms associated with Fbw7 loss may result from activation of any (or all) of these oncogenic targets. We constructed a knockin mouse in which the wild-type cyclin E gene was precisely replaced with one encoding a nonphosphorylatable residue, alanine, at the critical 393 position (equivalent to T380 in human cyclin E). This murine model has two important features: (1) cyclin E is resistant to Fbw7-mediated degradation but is transcribed at normal levels from its endogenous transcription unit; and (2) the role of disabled cyclin E turnover is separated from the activities of other oncogenic Fbw7 substrates caused by mutations within Fbw7 itself. We created a gene-targeting construct by using site-directed mutagenesis to change threonine 393 in exon 11 of the murine cyclin E gene to alanine, and by placing a Neor selectable marker flanked by LoxP sites into the intron between exons 10 and 11 and the gene encoding diphtheria toxin (DTA) upstream of exon1 (Figure 1). After transfection of embryonic stem (ES) cells and selection for G418 resistance, the precise replacement of the endogenous cyclin E gene by the cyclin ET393A allele was confirmed by PCR, Southern blot hybridization, and then direct genomic DNA sequencing to demonstrate the presence of the T393A mutation (Figure 1 and data not shown). Chimeric mice that demonstrated germline transmission of the cyclin ET393A allele were generated from two such ES cell clones. To eliminate the Neor marker, cyclin ET393A mice were mated with MORE mice, in which Cre recombinase has been targeted to the Mox-2 locus and is therefore expressed early germ cells (Tallquist and Soriano, 2000Tallquist M.D. Soriano P. Epiblast-restricted Cre expression in MORE mice: a tool to distinguish embryonic vs. extra-embryonic gene function.Genesis. 2000; 26: 113-115Crossref PubMed Scopus (276) Google Scholar). Deletion of the Neor gene was monitored by PCR amplification and confirmed by direct DNA sequencing (Figure 1). Mice were then backcrossed into both 129/SV and C57/Bl6 backgrounds for five generations. Interbreeding of cyclin ET393A heterozygous mice led to the recovery of all expected genotypes with normal Mendelian ratios (Figure 1D). Cyclin ET393A homozygous mice were followed for 2 years; they displayed no gross phenotypic abnormalities and appeared indistinguishable from the wild-type littermates. Detailed histopathological analysis revealed normal morphogenesis in all tissues examined. The only significant abnormality was a mild to moderate splenomegaly (1–2× weight) with mild extramedullary hematopoiesis. The cyclin ET393A mice displayed a normal life span with no significant predilection for hyperproliferative lesions or tumors. The effect of the cyclin ET393A mutation on the expression of endogenous cyclin E, its associated kinase activity, and cell cycle regulation was studied in endogenous mouse tissues and embryonic fibroblasts (MEFs) isolated from day 12–14 embryos. The cyclin E protein was readily detected in extracts prepared from the spleen and thymus from 2- to 4-month-old mice, tissues with many proliferating cells (Figure 2A ). However, the level of cyclin E and its associated kinase activity in the nonproliferative tissues from 2- to 4-month-old mice including lung, liver, and kidney was below the limit of detection (data not shown). Most strikingly, the abundance of the cyclin E protein in the splenic extracts from cyclin ET393A mice consistently showed a 5- to 10-fold increase compared to age-matched wild-type controls, while a more limited 2- to 3-fold elevation in the level of cyclin E was detected in cyclin ET393A thymic extracts. The amount of cyclin E-associated kinase activity closely paralleled the amount of cyclin E protein (Figure 2A). Consistent with the increase in cyclin E protein levels, freshly isolated ET393A splenocytes showed a 4-fold increase in the number of cycling cells compared to matched wild-type splenocytes (Figure 2B). Histologic examination of the corresponding splenic sections, performed to rule out an occult malignancy, showed only a mild increase in extramedullary hematopoiesis. Fractionation of the spleen into populations enriched for either B or T lymphocytes showed that cyclin ET393A was elevated equally in both compartments (data not shown). A small increase in cyclin E protein abundance and its associated kinase activity (1.5–2.2×) was detected in asynchronously proliferating heterozygous and homozygous cyclin ET393A MEFs (Figure 2C). This appeared to be due to a small increase in the half-life of cyclin ET393A (5 hr) in comparison to wild-type cyclin E (4 hr), as determined both by 35S pulse-chase measurement and by cycloheximide-mediated inhibition of protein synthesis (Figures 2D and 2E), whereas the amount of the cyclin ET393A mRNA was unchanged compared to wild-type cyclin E (data not shown). The surprisingly small effect of the T393A mutation on cyclin E stability in MEFs is explained by the assembly of most cyclin E protein into catalytically inactive complexes with the CDK inhibitor p21Cip1, which stabilizes cyclin E through the inhibition of its associated kinase activity, and is addressed in greater detail below (Clurman et al., 1996Clurman B.E. Sheaff R.J. Thress K. Groudine M. Roberts J.M. Turnover of cyclin E by the ubiquitin-proteasome pathway is regulated by cdk2 binding and cyclin phosphorylation.Genes Dev. 1996; 10: 1979-1990Crossref PubMed Scopus (402) Google Scholar, Welcker et al., 2003Welcker M. Singer J. Loeb K.R. Grim J. Bloecher A. Gurien-West M. Clurman B.E. Roberts J.M. Multisite phosphorylation by Cdk2 and GSK3 controls cyclin E degradation.Mol. Cell. 2003; 12: 381-392Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). The stability of cyclin ET393A was still regulated by ubiquitin-dependent degradation, since proteasome inhibition increased the half-life of cyclin ET393A from 5 hr to greater than 8 hr (data not shown). The modestly increased amount of cyclin ET393A was not associated with a consistent change in the distribution of MEFs into the G1, S, or G2/M compartments of the cell cycle (data not shown). We also examined the expression and activity of cyclin E in early passage MEFs that were synchronized by serum starvation/contact inhibition and then released into normal growth medium (Figure 3A ). Note that, in contrast to similar experiments performed using human cell lines, the amount of cyclin E protein in primary MEFs was close to invariant throughout the cell cycle, with a small peak in S phase. Cyclin E-associated kinase activity was more periodic than its overall protein abundance and was limited to late G1 and S phase (Figures 3A and 3B). The cyclin ET393A protein was more abundant, and its associated kinase activity was both higher and somewhat less periodic than the wild-type cyclin E protein and kinase activity. Parallel cultures were pulse labeled with BrdU and harvested for flow cytometry to determine the effect of the T393A mutation on progression through the cell cycle. In these early passage MEFs, the premature activation of cyclin ET393A-associated kinase activity did not have a reproducible effect on the rate at which cells completed G1 and entered S phase (Figure 3C). The elevated level of cyclin ET393A detected in splenic extracts suggested that loss of the T393 phosphorylation might have a significant effect on lymphocyte activation and proliferation. Resting T lymphocytes expressed barely detectable levels of cyclin E. Following activation, the level of cyclin ET393A increased earlier and to a greater extent than wild-type protein (Figure 3D). This resulted in an increase in the percentage of cells activated by a mitogenic stimulus (IL-2 receptor expression) and in the rate of cell proliferation (Figures 3E and 3F). These observations were consistent with the splenomegaly found in cyclin ET393A mice. Cyclin ET393A had a much more significant effect on cell proliferation of MEFs immortalized by a 3T3 protocol. The proliferation of control and cyclin ET393A MEFs entered a plateau between passages 5 and 10. In most experiments, the cyclin ET393A MEFs resumed exponential proliferation between passages 15 and 25, while the control cells frequently remained in the plateau phase until passages 25–30. A representative result is shown (Figure 4A ). This escape from senescence is a stochastic process with considerable variability among genetically identical clones of fibroblasts. Nevertheless, the cyclin ET393A fibroblasts typically immortalized earlier than control cells. After immortalization, cyclin ET393A MEFs grew faster and to a 2-fold greater saturation density (data not shown) than the immortalized control MEFs. The amount of cyclin E protein was also considerably greater in the cyclin ET393A immortalized MEFs compared to control immortalized MEFs (Figure 4B), and this was associated with an earlier entry into S phase in synchronized cells (Figure 4C). Our results suggested that the biological effects of cyclin ET393A were being constrained in early passage cells by a factor(s) that was lost upon immortalization. One candidate was the CDK inhibitor p21Cip1, which has previously been shown to limit the effects of misregulated cyclin E in transfected cells (Minella et al., 2002Minella A.C. Swanger J. Bryant E. Welcker M. Hwang H. Clurman B.E. p53 and p21 form an inducible barrier that protects cells against cyclin E-cdk2 deregulation.Curr. Biol. 2002; 12: 1817-1827Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). In the immortalized cells described above, p21 levels were reduced (data not shown), presumably as a secondary effect of p53 inactivation. To investigate the role of p21 in controlling cyclin E activity, we crossed the ET393A allele into a p21 null background. In most tissues, the amount of cyclin ET393A and its associated kinase activity were similar in cyclin ET393A and cyclin ET393A p21−/− mice (Figure 2A). In early passage MEFs, however, the loss of p21 had a significant impact on the abundance and activity of cyclin ET393A relative to wild-type cyclin E. As previously reported, cyclin E protein levels were greatly reduced in p21 null MEFs, and its half-life was reduced to 2 hr (Figure 4, Figure 2) (Welcker et al., 2003Welcker M. Singer J. Loeb K.R. Grim J. Bloecher A. Gurien-West M. Clurman B.E. Roberts J.M. Multisite phosphorylation by Cdk2 and GSK3 controls cyclin E degradation.Mol. Cell. 2003; 12: 381-392Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). This was thought to be caused by an increase in CDK2-dependent phosphorylation of cyclin E, and an increase in its rate of degradation by the T393-dependent pathway. Consistent with this idea, the stability and abundance of cyclin ET393A was largely unaffected by the loss of p21 (Figure 4, Figure 2), and as a consequence it was both more stable (Figures 2D and 2E) and more abundant (Figures 4D and 4E) than wild-type cyclin E in the p21 null background. The increased amount of cyclin ET393A in p21 null MEFs correlated with both an increased amount of cyclin E-associated kinase activity (Figures 4E and 4F) and increased rate of progression from quiescence to S phase (Figure 4G). Note that the cyclin ET393A p21−/− cells remained in S phase with a high percentage (60%) of BrdU-labeled cells at late time points, while the number of BrdU-positive p21−/− fibroblasts started to decline. This pattern suggested that the cyclin ET393A induced both early entry into and delayed progression through S phase in a p21 null background, similar to what has been seen in cells overexpressing wild-type cyclin E (Ohtsubo and Roberts, 1993Ohtsubo M. Roberts J.M. Cyclin-dependent regulation of G1 in mammalian fibroblasts.Science. 1993; 259: 1908-1912Crossref PubMed Scopus (652) Google Scholar, Minella et al., 2002Minella A.C. Swanger J. Bryant E. Welcker M. Hwang H. Clurman B.E. p53 and p21 form an inducible barrier that protects cells against cyclin E-cdk2 deregulation.Curr. Biol. 2002; 12: 1817-1827Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, Ekholm-Reed et al., 2004Ekholm-Reed S. Mendez J. Tedesco D. Zetterberg A. Stillman B. Reed S.I. Deregulation of cyclin E in human cells interferes with prereplication complex assembly.J. Cell Biol. 2004; 165: 789-800Crossref PubMed Scopus (219) Google Scholar). In summary, our data showed that induction of p21Cip1 could suppress the effect of the cyclin ET393A mutation. When p21 expression was reduced, either indirectly during spontaneous immortalization or by direct genetic targeting, this phenotypic suppression was lost, resulting in greater cyclin E levels, higher amounts of cyclin E-associated kinase activity, and aberrant timing of S phase initiation and completion. In contrast to cultured fibroblasts, the level of p21 in wild-type splenic extracts or resting splenocytes was undetectable prior to cell activation (data not shown). Moreover, the level of p21 expressed in activated wild-type splenocytes was 5- to 10-fold lower than that in proliferating MEFs. The preferential effect of cyclin ET393A on splenocytes of wild-type mice was therefore consistent with our data showing that the consequences of this mutation were increased in p21-deficient cells. Whereas cyclin ET393A slightly increased the rate of proliferation of early passage control MEFs, it significantly inhibited proliferation of p21 null cells (Figures 5A and 5B ). In contrast, cyclin ET393A significantly increased the proliferation rate of p53 null MEFs (cyclin ET393A p53−/−), thereby recapitulating the phenotype that we observed in spontaneously immortalized cells (Figure 5C). Thus, decreased p21 expression removed an intrinsic constraint on cyclin E activity, but this was associated with enhanced cell proliferation only if p53 was inactive. We next looked at long-term growth and senescence in a 3T3 protocol (Figure 5D). p21 null MEFs underwent a very transient senescence with minimal morphologic changes. In contrast, p21 null cells expressing cyclin ET393A showed a pronounced defect in proliferation rate and grossly altered cell morphology, from which they did not recover for at least 15 passages. The slow growth of cyclin ET393A p21−/− MEFs was associated with the accumulation of DNA damage. Phosphorylation of p53 on serine 15 is a sensitive indicator of DNA damage (Giaccia and Kastan, 1998Giaccia A.J. Kastan M.B. The complexity of p53 modulation: emerging patterns from divergent signals.Genes Dev. 1998; 12: 2973-2983Crossref PubMed Scopus (1142) Google Scholar). Asynchronously proliferating cyclin ET393A p21 null fibroblasts had an elevated amount of serine 15-phosphorylated p53 compared to p21 null fibroblasts (Figure 5E, compare lanes 1 and 3). Following irradiation, the level of p53 phosphorylated on serine 15 was elevated to a similar extent in both genotypes (Figure 5E, compare lan" @default.
• W2034151760 created "2016-06-24" @default.
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• W2034151760 creator A5036933165 @default.
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• W2034151760 date "2005-07-01" @default.
• W2034151760 modified "2023-10-16" @default.
• W2034151760 title "A mouse model for cyclin E-dependent genetic instability and tumorigenesis" @default.
• W2034151760 cites W1538085254 @default.
• W2034151760 cites W1569755992 @default.
• W2034151760 cites W1611210979 @default.
• W2034151760 cites W1868808920 @default.
• W2034151760 cites W1902628090 @default.
• W2034151760 cites W1966383348 @default.
• W2034151760 cites W1971254046 @default.
• W2034151760 cites W1981849277 @default.
• W2034151760 cites W1984012038 @default.
• W2034151760 cites W1989382065 @default.
• W2034151760 cites W1995788690 @default.
• W2034151760 cites W1997643531 @default.
• W2034151760 cites W1998964174 @default.
• W2034151760 cites W2017752436 @default.
• W2034151760 cites W2024466111 @default.
• W2034151760 cites W2027451946 @default.
• W2034151760 cites W2029620430 @default.
• W2034151760 cites W2041694710 @default.
• W2034151760 cites W2044057392 @default.
• W2034151760 cites W2068026673 @default.
• W2034151760 cites W2071152209 @default.
• W2034151760 cites W2072659475 @default.
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• W2034151760 cites W2095370504 @default.
• W2034151760 cites W2099524972 @default.
• W2034151760 cites W2102482497 @default.
• W2034151760 cites W2111486924 @default.
• W2034151760 cites W2113273914 @default.
• W2034151760 cites W2121619780 @default.
• W2034151760 cites W2126344344 @default.
• W2034151760 cites W2126423401 @default.
• W2034151760 cites W2138779676 @default.
• W2034151760 cites W2140834174 @default.
• W2034151760 cites W2152107294 @default.
• W2034151760 cites W2155881657 @default.
• W2034151760 cites W2156299289 @default.
• W2034151760 cites W2161135924 @default.
• W2034151760 cites W2167912686 @default.
• W2034151760 cites W2169281107 @default.
• W2034151760 cites W2169413646 @default.
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